The formation of bone and the regulation of bone formation has been an area of intense scientific interest and investigation for over twenty years. In part, such interest stems from a desire to therapeutically treat pathological disorders and diseases of bone such as those clinical conditions identified as Paget's disease, osteogenesis imperfecta, renal osteodystrophy, osteomalacias, osteopetrosis, and osteoporosis [Quelch et al., Calcif. Tissue Int. 36:545-549 (1984)]. Other investigators are primarily concerned with those alterations and changes of human bone which occur with increasing age, such changes being deemed normal although often debilitating to the individual [Dickson and Bagga, Conn. Tissue Res. 14:77-85 (1985)]. Regardless of the particular motive, it has become increasingly clear that the formation of bone and the regulation of bone formation must be viewed and understood at three individual levels: the tissue level; the cellular level; and the molecular matrix level [Raisz and Kream, N.E.J. Med. 309:29-35 and 83-89 (1983)]. At the tissue level, the concept of coupling of osteoclastic resorption and osteoblastic formation as bone remodeling units has become the central focus and controlling principle of bone metabolism [Harris and Heaney, N. Engl. J. Med. 280:253 (1969); Baylink and Liu, J. Periodontol. 50:43 (1979); Farley and Baylink, Trans. Assoc. Am. Physc. 94:80 (1981)]. In the adult skeleton, bone remodeling involves a coupled sequence of resorption followed by new bone formation which occurs at different sites to produce changes in size or shape and to maintain the mechanical strength of bone. This process is particularly important in the skeletal response to changes in mechanical stress and are, presumably, regulated locally rather than by systemic hormones. Because of these coupled processes, the absolute rates of bone resorption and formation in the skeleton are large relative to the net change in skeletal mass during normal aging. The overall effect, nevertheless, is that net bone mass increases during the first two decades of life and decreases after the third or fourth decade.
The cellular level of bone formation requires an understanding of the origin and fate of osteoblasts [Owen, M., Calcif. Tissue Res. 25:205-207 (1978)]. Each osteoblast carries out a cycle of matrix synthesis after which it becomes either buried as an internal osteocyte or remains as a surface but inactive osteocyte or resting osteoblast. The cellular level of control focuses on the premise that new bone formation and regulation is the result of activating resting osteoblasts or the proliferation and subsequent differentiation of new osteoblasts. Once activation occurs, it is the rate at which each osteoblast produces bone matrix or the duration of bone matrix synthesis which is the regulatory mechanism of control. It is widely believed, but not yet proven, that osteoblasts have a direct role in the regulation of bone mineralization. The fact that newly formed bone matrix does not mineralize immediately indicates that the matrix must undergo some changes before mineralization can occur [Raisz and Kream, N. Eng. J. Med. 309:29-35 (1983)].
The molecular matrix level of understanding and investigation focuses upon the minerals and proteins comprising bone itself. The minerals in the bone matrix are calcium and phosphate which typically take the form of hydroxylapatite [Termine, J. D., Clin. Orthop. 85:207-241 (1972); Blumenthal et al., Calcif. Tiss. Res. 18:81-90 (1975)]. The primary protein of the bone matrix is collagen which is secreted as procollagen by osteoblasts and assembled extracellularly into fibrils stabilized by intramolecular and intermolecular cross-linkages. In addition to collagen, the bone matrix comprises other protein secretions of osteoblasts. These include sialoprotein [Herring, G. M., Calcif. Tiss. Res. 24:29-36 (1977)]; osteocalcin [Poser et al., J. Biol. Chem. 255:8685-8691 (1980)]; and osteonectin [Termine et al., J. Biol. Chem. 256:10403-10408 (1981)]. The role of these noncollagenous proteins in normal and pathological human bone remains an area of considerable research [Quelch et al., Calcif. Tiss. Int. 36:545-549 (1984)]. Bone matrix also contains several plasma proteins as component compositions. These include serum albumin and .alpha..sub.2 HS-glycoprotein. Of all the compositions comprising the molecular matrix of bone, the presence of the .alpha..sub.2 HS-glycoprotein in the bone matrix remains a continuing mystery.
.alpha..sub.2 HS-glycoprotein was first isolated from normal human serum more than twenty-five years ago [Schmid and Burgi, Biochim. Biophys. Acta 47:440-453 (1961)]. This compound is a glycosylated, sulfate containing human serum protein consisting of two disulfide-linked polypeptide chains whose total molecular weight is approximately 50,000 daltons; the entirety of the amino acid sequences in each polypeptide chain has been established [Gejyo and Schmid, Biochim. Biophys. Acta 671:78-84 (1981); Gejyo et al., J. Biol. Chem. 258:496-497 (1983); Yoshioka et al., J. Biol. Chem. 261:1665-1676 (1986)]. The .alpha..sub.2 HS-glycoprotein is a negative acute phase reactant which is synthesized in the liver and displays genetic polymorphism [Lebreton et al., J. Clin. Invest. 64:1118-1129 (1979); Triffitt et al., Nature 262:226-227 (1967); Anderson et al., Proc. Natl. Acad. Sci. 74:5421-5425 (1977)]. Normal human serum levels of .alpha..sub.2 HS-glycoprotein are in the range of 600-660 micrograms per milliliter (hereinafter ".mu.g/ml") of serum [Dickson et al., Calcif. Tiss. Res. 35:16-20 (1983)] with the highest concentrations being found in young adults. Curiously, the concentration of .alpha..sub.2 HS-glycoprotein is reduced in humans afflicted with active Paget's disease [Ashton et al., Clin. Sci. 58:435-438 (1980)]; in persons having various solid tumors [Bradley et al., Cancer 40:2264-2272 (1977); Baskies et al., Cancer 45:3050-3060 (1980)]; and in individuals suffering from multiple myeloma [Crawford, S. M., Br. J. Cancer 49:813-815 (1984); Wiedermann et al., Neoplasma 25:189-196 (1978)]. This plasma protein is present in normal human cortical bone where it is concentrated approximately 140 fold with respect to other plasma proteins [Ashton et al., Calcif. Tiss. Res. 22:27-33 (1976); Triffitt et al., Calcif. Tiss. Res. 26:155-161 (1978); Quelch et al., Calcif. Tiss. Int. 36:545-549 (1984)]. Moreover, the concentration of .alpha..sub.2 HS-glycoprotein varies as a function of age with the concentration in fetal bone being more than ten times greater than the concentration in adult bone [Wilson et al., Calcif. Tiss. Res. 22: (Suppl.):458 (1977); Dickson and Bagga, Conn. Tiss. Res. 14:77-85 (1985)]. It is generally believed that .alpha..sub.2 HS-glycoprotein becomes concentrated in the bone matrix during mineralization due to its affinity for hydroxylapatite or calcium [Triffitt et al., Nature 262:226-227 (1967); Dickson et al., Nature 256:430-432 (1974)].
Despite this abundance of knowledge and data regarding .alpha..sub.2 HS-glycoprotein, the true role and biological function of this protein, if any, is yet to be established. As part of its observed properties, .alpha..sub.2 HS-glycoprotein is said to be an opsonin [Van Oss et al., Immunol. Commun. 3:329-335 (1974)]; to bind barium ions [Schmid and Burgi, Biochim. Biophys. Acta 47:440-453 (1961)]; to bind calcium ions [Ashton et al., Calcif. Tiss. Res. 22:27-33 (1976); Triffitt et al., Nature 262:226-227 (1976)]; to adhere to DNA [Lewis and Andre, FEBS Lett. 92:211-213 (1978)]; and to promote endocytosis by macrophages [Lewis and Andre, Immunology 39:317-322 (1980)]. Despite these reports, it is generally accepted in this scientific community that the biological function and true role of .alpha..sub.2 HS-glycoprotein in bone formation and bone regulation remains unknown and unappreciated to date.